Cellular Modifications Research Articles

ALCIAN BLUE pH;

Background: Alcian blue (AB) is a cationic dye that stains mucins especially acidic mucins into varying shades of blue colour depending on the pH of the dye. GIT comprises of three main portions, fore-, mid- and hind-gut, which develops from different embryological areas. Their epithelial lining presents with varying degree of diversity including their secretions. Studies have shown that there is a tendency of cellular modification (physical and chemical) including secretions during tumour morphogenesis. With recent increase in GIT tumours especially epithelial tumours, Alcian Blue staining of mucins produced by these tumours at different pH more than conventional pH might give valuable information on the property of these tumours. Setting: The study was conducted in the Histopathology and Morbid Anatomy unit, department of Laboratory services, Muhimbili National Hospital, Tanzania. Study design: This was a hospital based retrospective study, in which archival data and blocks were retrieved. Objective: To determine the effect of pH and pattern of Alcian blue staining on primary GIT epithelial tumours tumours. Materials and Methods: Information on patients were obtained from cancer registry and patient files. Paraffin blocks were retrieved from archive, sections were cut using rotary microtome at 3μm (SAKURA). Haematoxylin and Eosin (H&E) and AB staining at pH 1, 1.5, 2 and 2.5 was done for each case and control. H&E slides were reviewed for confirmation of the diagnosis primary epithelial tumours and AB stained slides were evaluated for staining reaction and graded. Results: Out of 87 GIT primary epithelial tumours which were evaluated, AB staining was positive in 21 (24.1%) cases, the majority of these (11 (52.4%)) were from hindgut. Positive AB staining of GIT epithelial tumours increased as the tumour became more differentiated irrespective of location. Majority of tumours with positive AB staining was observed at pH 2 in GIT epithelial tumours as opposed to the conventional pH of 1 and 2.5 respectively. Conclusions and recommendation: The majority of primary GIT epithelial tumours stained positively at pH 2 irrespective of the location. However tumour differentiation influenced AB staining whereby well-differentiated tumours were mostly positively stained. It is recommended that AB at pH 2 should be applied when staining GIT epithelial tumours rather than conventional pH of 1 and 2.5. However the degree of differentiation should be considered since poorly differentiated tumours are likely to give negative results with AB staining.

In planta processing and glycosylation of a nematode CLAVATA3/ENDOSPERM SURROUNDING REGION-like effector and its interaction with a host CLAVATA2-like receptor to promote parasitism.

Like other biotrophic plant pathogens, plant-parasitic nematodes secrete effector proteins into host cells to facilitate infection. Effector proteins that mimic plant CLAVATA3/ENDOSPERM SURROUNDING REGION-related (CLE) proteins have been identified in several cyst nematodes, including the potato cyst nematode (PCN); however, the mechanistic details of this cross-kingdom mimicry are poorly understood. Plant CLEs are posttranslationally modified and proteolytically processed to function as bioactive ligands critical to various aspects of plant development. Using ectopic expression coupled with nanoliquid chromatography-tandem mass spectrometry analysis, we show that the in planta mature form of proGrCLE1, a multidomain CLE effector secreted by PCN during infection, is a 12-amino acid arabinosylated glycopeptide (named GrCLE1-1Hyp4,7g) with striking structural similarity to mature plant CLE peptides. This glycopeptide is more resistant to hydrolytic degradation and binds with higher affinity to a CLAVATA2-like receptor (StCLV2) from potato (Solanum tuberosum) than its nonglycosylated forms. We further show that StCLV2 is highly up-regulated at nematode infection sites and that transgenic potatoes with reduced StCLV2 expression are less susceptible to PCN infection, indicating that interference of the CLV2-mediated signaling pathway confers nematode resistance in crop plants. These results strongly suggest that phytonematodes have evolved to utilize host cellular posttranslational modification and processing machinery for the activation of CLE effectors following secretion into plant cells and highlight the significance of arabinosylation in regulating nematode CLE effector activity. Our finding also provides evidence that multidomain CLEs are modified and processed similarly to single-domain CLEs, adding new insight into CLE maturation in plants.

Astrocytes alignment and reactivity on collagen hydrogels patterned with ECM proteins

To modulate the surface properties of collagen and subsequent cell–surface interactions, a method was developed to transfer protein patterns from glass coverslips to collagen type I hydrogel surfaces. Two proteins and one proteoglycan found in central nervous system extracellular matrix as well as fibrinogen were patterned in stripes onto collagen hydrogel and astrocytes were cultured on these surfaces. The addition of the stripe protein patterns to hydrogels created astrocyte layers in which cells were aligned with underlying patterns and had reduced chondroitin sulfate expression compared to the cells grown on collagen alone. Protein patterns were covalently cross-linked to the collagen and stable over four days in culture with no visible cellular modifications. The present method can be adapted to transfer other types of protein patterns from glass coverslips to collagen hydrogels.

Proteomics analysis of E-cadherin knockdown in epithelial breast cancer cells

E-cadherin is the core protein of the epithelial adherens junction. Through its cytoplasmic domain, E-cadherin interacts with several signaling proteins; among them, α- and β-catenins mediate the link of E-cadherin to the actin cytoskeleton. Loss of E-cadherin expression is a crucial step of epithelial-mesenchymal transition (EMT) and is involved in cancer invasion and metastatization. In human tumors, down-regulation of E-cadherin is frequently associated with poor prognosis. Despite the critical role of E-cadherin in cancer progression, little is known about proteome alterations linked with its down-regulation. To address this point, we investigated proteomics, biophysical and functional changes of epithelial breast cancer cell lines upon shRNA-mediated stable knockdown of E-cadherin expression (shEcad). shEcad cells showed a distinct proteomic signature including altered expression of enzymes and proteins involved in cytoskeletal dynamic and migration. Moreover, these results suggest that, besides their role in mechanical adhesion, loss of E-cadherin expression may contribute to cancer progression by modifying a complex network of pathways that tightly regulate fundamental processes as oxidative stress, immune evasion and cell metabolism. Altogether, these results extend our knowledge on the cellular modifications associated with E-cadherin down-regulation in breast cancer cells.

Removal of a small C-terminal region of JCV and SV40 large T antigens has differential effects on transformation

The large T antigen (LT) protein of JCV and SV40 polyomaviruses is required to induce tumors in rodents and transform cells in culture. When both LTs are compared side-by-side in cell culture assays, SV40 shows a more robust transformation phenotype even though the LT sequences are highly conserved. A complete understanding of SV40׳s enhanced transforming capabilities relative to JCV is lacking. When the least conserved region of the LT proteins, the variable linker and host range region (VHR), was removed, changes in T antigen expression and cellular p53 post-translational modifications occurred, but interaction with the pRB pathway was unaffected. Transformation assessed by growth in low serum was reduced after VHR truncation of the SV40, but not the JCV, T antigen. Conversely, anchorage independent transformation was enhanced only by truncation of the JCV VHR. This is the first report to link the SV40 or JCV VHR region to transformation potential.

Cellular proteolytic modification of tumor-suppressor CYLD is critical for the initiation of human T-cell acute lymphoblastic leukemia.

There exists a general recognition of the fact that post translational modification of CYLD protein through proteolytic cleavage by MALT-1 results in sustained cellular NF-kB activity which is conspicuously found to be associated with cancer in general and hematological malignancies in particular. The present study was directed to understand the contribution of MALT-1 and deubiquitinase CYLD to the initiation of T-cell acute lymphoblastic leukemia (T-ALL). Such a study revealed for the first time that the 35kDa CYLD cleaved factor generated by MALT-1 mediated proteolytic cleavage was conspicuously present in human T- ALL subjects of pediatric age group. Further, over-expression of this 35kDa CYLD factor within normal human peripheral blood mononuclear cells had the inherent capacity to program the genome of these cells resulting in T-cell lineage ALL. Based upon these results, we propose that MALT1 inhibitors may be of crucial importance in the treatment of T-ALL subjects of pediatric age group.

Impaired liver function in Xenopus tropicalis exposed to benzo[a]pyrene: transcriptomic and metabolic evidence.

BackgroundDespite numerous studies suggesting that amphibians are highly sensitive to cumulative anthropogenic stresses, the role pollutants play in the decline of amphibian populations remains unclear. Amongst the most common aquatic contaminants, polycyclic aromatic hydrocarbons (PAHs) have been shown to induce several adverse effects on amphibian species in the larval stages. Conversely, adults exposed to high concentrations of the ubiquitous PAH, benzo[a]pyrene (BaP), tolerate the compound thanks to their highly efficient hepatic detoxification mechanisms. Due to this apparent lack of toxic effect on adults, no studies have examined in depth the potential toxicological impact of PAH on the physiology of adult amphibian livers. This study sheds light on the hepatic responses of Xenopus tropicalis when exposed to high environmentally relevant concentrations of BaP, by combining a high throughput transcriptomic approach (mRNA deep sequencing) and a characterization of cellular and physiological modifications to the amphibian liver.ResultsTranscriptomic changes observed in BaP-exposed Xenopus were further characterized using a time-dependent enrichment analysis, which revealed the pollutant-dependent gene regulation of important biochemical pathways, such as cholesterol biosynthesis, insulin signaling, adipocytokines signaling, glycolysis/gluconeogenesis and MAPK signaling. These results were substantiated at the physiological level with the detection of a pronounced metabolic disorder resulting in a possible insulin resistance-like syndrome phenotype. Hepatotoxicity induced by lipid and cholesterol metabolism impairments was clearly identified in BaP-exposed individuals.ConclusionsOur data suggested that BaP may disrupt overall liver physiology, and carbohydrate and cholesterol metabolism in particular, even after short-term exposure. These results are further discussed in terms of how this deregulation of liver physiology can lead to general metabolic impairment in amphibians chronically exposed to contaminants, thereby illustrating the role xenobiotics might play in the global decline in amphibian populations.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-666) contains supplementary material, which is available to authorized users.

Chasing Phosphoarginine Proteins: Development of a Selective Enrichment Method Using a Phosphatase Trap

Arginine phosphorylation is an emerging post-translational protein modification implicated in the bacterial stress response. Although early reports suggested that arginine phosphorylation also occurs in higher eukaryotes, its overall prevalence was never studied using modern mass spectrometry methods, owing to technical difficulties arising from the acid lability of phosphoarginine. As shown recently, the McsB and YwlE proteins from Bacillus subtilis function as a highly specific protein arginine kinase and phosphatase couple, shaping the phosphoarginine proteome. Using a B. subtilis ΔywlE strain as a source for arginine-phosphorylated proteins, we were able to adapt mass spectrometry (MS) protocols to the special chemical properties of the arginine modification. Despite this progress, the analysis of protein arginine phosphorylation in eukaryotes is still challenging, given the great abundance of serine/threonine phosphorylations that would compete with phosphoarginine during the phosphopeptide enrichment procedure, as well as during data-dependent MS acquisition. We thus set out to establish a method for the selective enrichment of arginine-phosphorylated proteins as an initial step in the phosphoproteomic analysis. For this purpose, we developed a substrate-trapping mutant of the YwlE phosphatase that retains binding affinity toward arginine-phosphorylated proteins but cannot hydrolyze the captured substrates. By testing a number of active site substitutions, we identified a YwlE mutant (C9A) that stably binds to arginine-phosphorylated proteins. We further improved the substrate-trapping efficiency by impeding the oligomerization of the phosphatase mutant. The engineered YwlE trap efficiently captured arginine-phosphorylated proteins from complex B. subtilis ΔywlE cell extracts, thus facilitating identification of phosphoarginine sites in the large pool of cellular protein modifications. In conclusion, we present a novel tool for the selective enrichment and subsequent MS analysis of arginine phosphorylation, which is a largely overlooked protein modification that might be important for eukaryotic cell signaling.

Molecular targets of chromatin repressive mark H3K9me3 in primate progenitor cells within adult neurogenic niches

Histone 3 Lysine 9 (H3K9) methylation is known to be associated with pericentric heterochromatin and important in genomic stability. In this study, we show that trimethylation at H3K9 (H3K9me3) is enriched in an adult neural stem cell niche- the subventricular zone (SVZ) on the walls of the lateral ventricle in both rodent and non-human primate baboon brain. Previous studies have shown that there is significant correlation between baboon and human regarding genomic similarity and brain structure, suggesting that findings in baboon are relevant to human. To understand the function of H3K9me3 in this adult neurogenic niche, we performed genome-wide analyses using ChIP-Seq (chromatin immunoprecipitation and deep-sequencing) and RNA-Seq for in vivo SVZ cells purified from baboon brain. Through integrated analyses of ChIP-Seq and RNA-Seq, we found that H3K9me3-enriched genes associated with cellular maintenance, post-transcriptional and translational modifications, signaling pathways, and DNA replication are expressed, while genes involved in axon/neuron, hepatic stellate cell, or immune-response activation are not expressed. As neurogenesis progresses in the adult SVZ, cell fate restriction is essential to direct proper lineage commitment. Our findings highlight that H3K9me3 repression in undifferentiated SVZ cells is engaged in the maintenance of cell type integrity, implicating a role for H3K9me3 as an epigenetic mechanism to control cell fate transition within this adult germinal niche.

An imbalance between Beclin-1 and p62 expression promotes the proliferation of myeloma cells through autophagy regulation

Autophagy occurs in tumor cells acquiring cytotoxic drug resistance and its activation may impair their susceptibility to apoptosis in response to apoptogen agents. We investigated the pro-apoptotic effect of dexamethasone (Dex) on MMcell lines (U266, INA-6, LR5-8226, LIG, and MCC2) and primary malignant plasma cells from naïve and refractory/relapsed patients. We evaluated the transcriptional and ultrastructural events leading to autophagy by measuring Beclin-1 and p62 levels and transmission electronic microscopy. Autophagy was inhibited by hydroxychloroquine (HCQ), whereas the ability of Dex-resistant MMcells to recover the susceptibility to apoptosis was measured. A direct relationship between autophagy and Beclin-1 or LC3/Atg8 levels was observed, whereas their mRNAs were inversely correlated to p62 expression. Starvation strongly activated autophagy by inducing cellular, transcriptional, and ultrastructural modifications that were reversed by HCQ. Taken together, these data suggest that autophagy is a potential mechanism leading to drug resistance in MM, and suggest Beclin-1 and p62 as early markers of cell susceptibility to apoptosis. The combination of HCQ with novel agents may thus be considered to improve the therapeutic response in relapsed/resistant MM patients.

Dinuclear berenil-platinum (II) complexes as modulators of apoptosis in human MCF-7 and MDA-MB231 breast cancer cells.

The metabolism of alkylating agents is accompanied by the generation of reactive oxygen species. The aim of this study was to treat estrogen receptor-positive and estrogen receptor-negative human breast cancer cells, MCF-7 and MDA-MB-231, respectively, with cisplatin and five different berenil-platinum (II) complexes, and then to investigate the oxidative modifications of DNA, lipid and protein, and to compare them with the profile of various pro- and antiapoptotic proteins. Changes in the levels of 8-hydroxy-2'-deoxyguanosine, 4-hydroxynonenal, carbonyl groups, dityrosine, active caspases 3, 8 and 9, as well as the expression of Bcl-2, Bax, cytochrome c, and p53 were subsequently examined. Activities of superoxide dismutase, catalase and glutathione, vitamin C levels were also investigated. Cellular reactions to cisplatin and the berenil-platinum (II) derivatives were more pronounced in MCF-7 cells as compared with the MDA-MB231 cells. Furthermore, the berenil-platinum (II) derivatives were found to be more effective than cisplatin. All of the complexes reduced the activity of superoxide dismutase and catalase, and also lowered the levels of non-enzymatic antioxidants. Increased level of lipid, protein as well as DNA damage markers was also observed after berenil-platinum (II) derivatives treatment. Similarly, the increase in the levels of the proapoptotic factors, were detected in MCF-7 and MDA-MB231 cells. Incubation of examined cells with the berenil-platinum (II) complexes also led to the increase in the levels of active caspases 3, 8 and 9. In conclusion, the results of the present study demonstrated that berenil-platinum (II) complexes more efficiently mediate cellular oxidative modifications and proapoptotic metabolism, particularly in MCF-7 cells, compared to cisplatin. Pt2(isopropylamine)4berenil2 and Pt2(piperidine)4(berenil)2 notably affected the cellular metabolism of estrogen-positive breast cancer cells. Thus, these complexes may be valuable for the design of additional anticancer drugs.

The role of hepatic lipids in hepatic insulin resistance and type 2 diabetes.

Non-alcoholic fatty liver disease and its downstream sequelae, hepatic insulin resistance and type 2 diabetes, are rapidly growing epidemics, which lead to increased morbidity and mortality rates, and soaring health-care costs. Developing interventions requires a comprehensive understanding of the mechanisms by which excess hepatic lipid develops and causes hepatic insulin resistance and type 2 diabetes. Proposed mechanisms implicate various lipid species, inflammatory signalling and other cellular modifications. Studies in mice and humans have elucidated a key role for hepatic diacylglycerol activation of protein kinase Cε in triggering hepatic insulin resistance. Therapeutic approaches based on this mechanism could alleviate the related epidemics of non-alcoholic fatty liver disease and type 2 diabetes.

The influence of cellular concrete surface modification on its physical and mechanical properties

Interaction of polyorganosiloxanes of different spatial structures with polymethylmethacrylate and epoxide resin in different ratios was investigated, namely double and triple systems were considered in the paper. The obtained systems were used in modifying the cellular concrete surface and identifying the main physical and mechanical properties such as surface adsorption, impregnation depth, water absorption, ultimate bending strength.Modifying the surface of cellular concrete was conducted to improve its performance properties since at the large number of open capillary pores during operation in outdoor conditions, products lose their basic feature − heat insulating. Also, modified products have a 5 times higher ultimate bending strength.As a result of studies, it was found that impregnations, based on silicone varnish KO-075 were the most effective modifying compositions. They united 2 principles at once: surface hydrophobization with its simultaneous strengthening. The obtained data are of great interest for both researchers and cellular concrete products manufacturers. Modifiedcellular concrete products can be used as supporting structures in low-rise construction that in turn will lead to saving energy and labor costs.

Mesenchymal Stem Cells from Rats with Chronic Kidney Disease Exhibit Premature Senescence and Loss of Regenerative Potential

Mesenchymal stem cell (MSC) transplantation has the potential for organ repair. Nevertheless, some factors might lessen the regenerative potential of MSCs, e.g. donor age or systemic disease. It is thus important to carefully assess the patient's suitability for autologous MSC transplantation. Here we investigated the effects of chronic kidney disease (CKD) on MSC function. We isolated bone marrow MSCs from remnant kidney rats (RK) with CKD (CKD-RK-MSC) and found signs of premature senescence: spontaneous adipogenesis, reduced proliferation capacity, active senescence-associated-β-galactosidase, accumulation of actin and a modulated secretion profile. The functionality of CKD-RK-MSCs in vivo was tested in rats with acute anti-Thy1.1-nephritis, where healthy MSCs have been shown to be beneficial. Rats received healthy MSCs, CKD-RK-MSC or medium by injection into the left renal artery. Kidneys receiving healthy MSCs exhibited accelerated healing of glomerular lesions, whereas CKD-RK-MSC or medium exerted no benefit. The negative influence of advanced CKD/uremia on MSCs was confirmed in a second model of CKD, adenine nephropathy (AD). MSCs from rats with adenine nephropathy (CKD-AD-MSC) also exhibited cellular modifications and functional deficits in vivo. We conclude that CKD leads to a sustained loss of in vitro and in vivo functionality in MSCs, possibly due to premature cellular senescence. Considering autologous MSC therapy in human renal disease, studies identifying uremia-associated mechanisms that account for altered MSC function are urgently needed.

Structural alterations of skeletal muscle in copd

Background: Chronic obstructive pulmonary disease (COPD) is a respiratory disease associated with a systemic inflammatory response. Peripheral muscle dysfunction has been well characterized in individuals with COPD and results from a complex interaction between systemic and local factors.Objective: In this narrative review, we will describe muscle wasting in people with COPD, the associated structural changes, muscle regenerative capacity and possible mechanisms for muscle wasting. We will also discuss how structural changes relate to impaired muscle function and mobility in people with COPD.Key Observations: Approximately 30–40% of individuals with COPD experience muscle mass depletion. Furthermore, muscle atrophy is a predictor of physical function and mortality in this population. Associated structural changes include a decreased proportion and size of type-I fibers, reduced oxidative capacity and mitochondrial density mainly in the quadriceps. Observations related to impaired muscle regenerative capacity in individuals with COPD include a lower proportion of central nuclei in the presence or absence of muscle atrophy and decreased maximal telomere length, which has been correlated with reduced muscle cross-sectional area. Potential mechanisms for muscle wasting in COPD may include excessive production of reactive oxygen species (ROS), altered amino acid metabolism and lower expression of peroxisome proliferator-activated receptors-gamma-coactivator 1-alpha mRNA. Despite a moderate relationship between muscle atrophy and function, impairments in oxidative metabolism only seems weakly related to muscle function.Conclusion: This review article demonstrates the cellular modifications in the peripheral muscle of people with COPD and describes the evidence of its relationship to muscle function. Future research will focus on rehabilitation strategies to improve muscle wasting and maximize function.

Karyopherins: potential biological elements involved in the delayed graft function in renal transplant recipients

BackgroundImmediately after renal transplantation, patients experience rapid and significant improvement of their clinical conditions and undergo considerable systemic and cellular modifications. However, some patients present a slow recovery of the renal function commonly defined as delayed graft function (DGF). Although clinically well characterized, the molecular mechanisms underlying this condition are not totally defined, thus, we are currently missing specific clinical markers to predict and to make early diagnosis of this event.MethodsWe investigated, using a pathway analysis approach, the transcriptomic profile of peripheral blood mononuclear cells (PBMC) from renal transplant recipients with DGF and with early graft function (EGF), before (T0) and 24 hours (T24) after transplantation.ResultsBioinformatics/statistical analysis showed that 15 pathways (8 up-regulated and 7 down-regulated) and 11 pathways (5 up-regulated and 6 down-regulated) were able to identify DGF patients at T0 and T24, respectively. Interestingly, the most up-regulated pathway at both time points was NLS-bearing substrate import into nucleus, which includes genes encoding for several subtypes of karyopherins, a group of proteins involved in nucleocytoplasmic transport. Signal transducers and activators of transcription (STAT) utilize karyopherins-alpha (KPNA) for their passage from cytoplasm into the nucleus. In vitro functional analysis demonstrated that in PBMCs of DGF patients, there was a significant KPNA-mediated nuclear translocation of the phosphorylated form of STAT3 (pSTAT3) after short-time stimulation (2 and 5 minutes) with interleukin-6.ConclusionsOur study suggests the involvement, immediately before transplantation, of karyopherin-mediated nuclear transport in the onset and development of DGF. Additionally, it reveals that karyopherins could be good candidates as potential DGF predictive clinical biomarkers and targets for pharmacological interventions in renal transplantation. However, because of the low number of patients analyzed and some methodological limitations, additional studies are needed to validate and to better address these points.

SUMOylation of nonstructural 5A protein regulates hepatitis C virus replication

Viruses exploit cellular SUMOylation machinery to favour their own propagation. We show that NS5A is a target protein of small ubiquitin-like modifier (SUMO) and is SUMOylated at lysine residue 348. We demonstrated that SUMOylation increased protein stability of NS5A by inhibiting ubiquitylation, and SUMOylation was also required for protein interaction with NS5B. These data imply that SUMO modification may contribute to HCV replication. Indeed, silencing of UBC9 impaired HCV replication in Jc1-infected cells, and HCV replication level was also significantly reduced in SUMO-defective subgenomic replicon cells. Taken together, these data indicate that HCV replication is regulated by SUMO modification of NS5A protein. We provide evidence for the first time that HCV exploits host cellular SUMO modification system to favour its own replication.

Obesity and Surgical Wound Healing: A Current Review

Objective. The correlation between obesity and deficient wound healing has long been established. This review examines the current literature on the mechanisms involved in obesity-related perioperative morbidity. Methods. A literature search was performed using Medline, PubMed, Cochrane Library, and Internet searches. Keywords used include obesity, wound healing, adipose healing, and bariatric and surgical complications. Results. Substantial evidence exists demonstrating that obesity is associated with a number of postoperative complications. Specifically in relation to wound healing, explanations include inherent anatomic features of adipose tissue, vascular insufficiencies, cellular and composition modifications, oxidative stress, alterations in immune mediators, and nutritional deficiencies. Most recently, advances made in the field of gene array have allowed researchers to determine a few plausible alterations and deficiencies in obese individuals that contribute to their increased risk of morbidity and mortality, especially wound complications. Conclusion. While the literature discusses how obesity may negatively affect health on various of medical fronts, there is yet to be a comprehensive study detailing all the mechanisms involved in obesity-related morbidities in their entirety. Improved knowledge and understanding of obesity-induced physiological, cellular, molecular, and chemical changes will facilitate better assessments of surgical risks and outcomes and create efficient treatment protocols for improved patient care of the obese patient population.

Sodium channels as gateable non-photonic sensors for membrane-delimited reactive species

Reactive oxygen species (ROS) and reactive oxygen intermediates (ROI) play crucial roles in physiological processes. While excessive ROS damages cells, small fluctuations in ROS levels represent physiological signals important for vital functions. Despite the physiological importance of ROS, many fundamental questions remain unanswered, such as which types of ROS occur in cells, how they distribute inside cells, and how long they remain in an active form. The current study presents a ratiometric sensor of intracellular ROS levels based on genetically engineered voltage-gated sodium channels (roNaV). roNaV can be used for detecting oxidative modification that occurs near the plasma membrane with a sensitivity similar to existing fluorescence-based ROS sensors. Moreover, roNaV has several advantages over traditional sensors because it does not need excitation light for sensing, and thus, can be used to detect phototoxic cellular modifications. In addition, the ROS dynamic range of roNaV is easily manipulated in real time by means of the endogenous channel inactivation mechanism. Measurements on ROS liberated from intracellular Lucifer Yellow and genetically encoded KillerRed have revealed an assessment of ROS lifetime in individual mammalian cells. Flashlight-induced ROS concentration decayed with two major time constants of about 10 and 1000ms.

Differential expression profiling of hypothalamus genes in laying period and ceased period Huoyan geese

The hypothalamus plays a central role in controlling poultry reproductive activity. To increase our understanding of genes involved in egg laying of Huoyan geese, gene profiles in the hypothalamus of laying period and ceased period Huoyan geese were investigated using suppression subtractive hybridization (SSH) method. A total of 95 differentially expressed sequence tags (ESTs), including 46 up-regulated and 49 down-regulated sequences showed homology to known genes of the non-redundant NCBI databases. Bioinformatic analysis demonstrated that these genes were mainly involved in anatomical structure development, signal transduction, cellular nitrogen compound metabolic process, biosynthetic process, cellular protein modification process, cell differentiation, transport, cell adhesion, and reproduction. Ten ESTs were selected for further analyses by quantitative real-time RT-PCR (qRT-PCR). Whose most part of results were consistent with the SSH results. Of note, AdipoR2, Nrg1, and NCAM1, which related with secretion of GnRH and other hormones, were identified to be differentially expressed between laying period and ceased period. These findings provided a new source for mining genes related to higher laying performance of Huoyan geese, which facilitate our understanding of the reproductive biology of the goose.